ABSTRACT
A "sandwich" fluorescence immunoassay is described which does not require the physical separation of bound from free label. Antibody coated microspheres, sample and fluorescent antibody are reacted together as in a conventional 'sandwich' immunoassay except that separation and washing steps are omitted. After the reaction is completed, the suspension is introduced directly into a flow cytometer equipped with a laser light source and both fluorescent and scattered light detection capabilities. By gating fluorescence light accumulation on scattered light pulses, particles associated fluorescence may be selectively measured. The system was evaluated in a model immunoassay for human immunoglobulin (hIgG), employing anti-hIgG coated microspheres (1--5 micrometer and 40--50 micrometer polyacrylamide beads and 30--40 micrometer dextran beads), fluorescein-labeled rabbit anti-hIgG and a Spectrum III flow cytometer. Sensitivities of 10 ng/ml and intra-assay precisions of 2--10% were achieved in a serum matrix. The approach potentially provides a general nonseparation immunoassay format for quantitatively measuring both small and large molecular weight soluble antigens, as well as cell surface antigens.
Subject(s)
Antigens, Surface/analysis , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunoglobulin G , MicrospheresABSTRACT
An improved radioimmunoassay technique for measuring serum thymopoietin has been developed which obviates the spurious displacements previously obtained with serum samples, and increases sensitivity to 20 pg. Key improvements involved further purification of labeled thymopoietin, sequential incubations to enhance sensitivity and the use of charcoal-treated serum blanks to render controls and unknown samples comparable to the standard curve.
Subject(s)
Radioimmunoassay/methods , Thymopoietins/blood , Thymus Hormones/blood , Animals , Cattle , Chromatography, Gel , Chromatography, Ion Exchange , Humans , Iodine Radioisotopes , MicrochemistryABSTRACT
A graphical procedure for determining the specific activity of radiolabeled ligands has been developed for use with radioimmunoassays. Although with this procedure we utilize the same experimental information required for displacement analysis, we are also able to determine both the specific activity and the binding constants of the labeled and unlabeled materials without assuming that these constants are equal; the concentration of antibody-binding sites can also be calculated. Thus, this graphical technique permits calculation of additional information without additional experimentation. We applied this procedure to the labeled materials used in a thymopoietin assay, testing two different preparations of radiolabeled material, and saw negligible differences between the two. The specific activity determined from the displacement analysis correlated well with that calculated by the graphical procedure.
